Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder

Abstract

Epithelial integrity in metazoan organs is maintained through the regulated proliferation and differentiation of organ-specific stem and progenitor cells. Although the epithelia of organs such as the intestine regenerate constantly and thus remain continuously proliferative, other organs, such as the mammalian urinary bladder, shift from near-quiescence to a highly proliferative state in response to epithelial injury. The cellular and molecular mechanisms underlying this injury-induced mode of regenerative response are poorly defined. Here we show in mice that the proliferative response to bacterial infection or chemical injury within the bladder is regulated by signal feedback between basal cells of the urothelium and the stromal cells that underlie them. We demonstrate that these basal cells include stem cells capable of regenerating all cell types within the urothelium, and are marked by expression of the secreted protein signal Sonic hedgehog (Shh). On injury, Shh expression in these basal cells increases and elicits increased stromal expression of Wnt protein signals, which in turn stimulate the proliferation of both urothelial and stromal cells. The heightened activity of this signal feedback circuit and the associated increase in cell proliferation appear to be required for restoration of urothelial function and, in the case of bacterial injury, may help clear and prevent further spread of infection. Our findings provide a conceptual framework for injury-induced epithelial regeneration in endodermal organs, and may provide a basis for understanding the roles of signalling pathways in cancer growth and metastasis.

Figure 1. Injury-induced proliferation and Hedgehog signalling in the bladder

a, UTI89 instillation induces proliferation of basal epithelial and stromal cells of the bladder. Ki67, Ck5 and laminin immunostaining highlight proliferation, basal epithelial cells and the basement membrane, respectively, in bladders 24 h after instillation of UTI89. Adjacent sections were 10 mm apart. b, Quantification of epithelial and stromal cell proliferation in response to bacterial injury. Ki67-positive cells are shown as a per cent of total 4′,6-diamidino-2-phenylindole (DAPI)-staining nuclei. c, Quantification of epithelial and stromal cell proliferation in response to chemical injury. Ki67-positive cells are shown as a per cent of total DAPI-staining nuclei 24 h after instillation of the indicated concentrations of PS. Note the absence of a proliferative response in the stroma. For panels b and c, data are from 3 bladders, 2 sections each, and are shown as mean ± s.e.m.; numerical data are in Supplementary Tables 1 and 2, respectively. d, Expression of eGFP in basal epithelial cells from a Shh-eGFP BAC transgenic mouse. e, Gli1-LacZ expression in the stromal compartment. Bladder sections from Gli1^LacZ/WT mice were co-stained with X-gal and anti-laminin. Scale bars in panels a, d and e represent 50 µm.

Figure 2. Shh-expressing basal cells repopulate the urothelium and form organoids in vitro

a, eGFP-marked Shh-expressing cells generate luminal cells positive for uroplakin 3. Shh^CreER/WT; R26^mTmG/WT mice were treated with TM and subjected to three rounds of injury. Uroplakin-3-positive and negative cells are denoted by white arrows and arrowheads, respectively. Dotted lines demarcate urothelium and stroma (L, lumen; S, stroma). b, Long-term regenerative capacity of eGFP-marked Shh-expressing cells. After TM injection and seven rounds of injury over 10 months, mG expression in bladder from Shh^CreER/WT; R26^mTmG/WT mice marks most urothelial cells. See Supplementary Fig. 2b for experimental schemes. c, Extended culture of Shh-expressing cells in Matrigel. Single eGFP-positive bladder cells isolated from TM-injected Shh^CreER/WT; R26^mTmG/WT mice and cultured in Matrigel for 5 weeks formed organoids. Dissociated cells from primary culture (P0) organoids also generated new organoids on subsequent passage (P1) in Matrigel culture. DIC, differential interference contrast. d, Confocal analysis of a bladder organoid. The organoid has multiple layers of epithelial cells with Ck5-expressing cells in the outer layer that contacts the extracellular matrix, and inner cells that line a luminal space and do not express Ck5. e, A section through the wall of an organoid grown in Matrigel and immunostained for Shh. Note eGFP expression in all cells, indicative of Shh expression in the cell initially cultured, but loss of Shh immunostaining from cells that are not in the outer layer. Scale bars represent 50 µm; asterisks denote organoid lumen.

Figure 3. Gli1 mediates injury-induced proliferation, restoration of urothelial integrity and reduction of infectious spread

a, Gli1 loss delays and attenuates proliferative response to bacterial injury. Bladders from wild-type or homozygous Gli1 mutant mice were analysed at the indicated times after UTI89 instillation by immunostaining for Ki67 and laminin. b, Quantification of Gli1 effect on epithelial and stromal cell proliferation. Ki67-positive cells are shown as a per cent of total DAPI-staining nuclei at the indicated times after UTI89 instillation in wild-type and Gli1 mutant bladders. c, Gli1 loss blocks the proliferative response of epithelial cells to chemical injury. Ki67-positive cells are shown as a per cent of total DAPI-staining nuclei 24 h after instillation of the indicated concentrations of PS. In panels b and c, data are shown as mean ± s.e.m. from 3 bladders, 2 sections each, and numerical data are shown in Supplementary Tables 4 and 6, respectively. d, Paracellular permeability in injured bladders from Gli1 mutant mice. Bladders from wild-type and Gli1 homozygotes were instilled with UTI89 and analysed at the times indicated after infection, with FITC-dextran instillation preceding bladder collection by 1.5 h. Dotted lines demarcate the border between urothelium and stroma. Note that normal reduced levels of paracellular permeability are restored by 24 h in wild-type but not Gli1 homozygotes. e, Infectious spread to kidneys is enhanced by Gli1 loss. Bacterial titres 24 h after UTI89 instillation were lower in bladders from Gli1 homozygotes as compared to wild-type (6.6 ± 3.27 × 10⁶ versus 3.2 ± 1.03 ± 10⁷ colony-forming units (c.f.u.); P < 0.05). In contrast, bacterial titres were significantly higher in kidneys from Gli1 homozygotes as compared to wild-type (2.59 ± 1.0 × 10⁵ versus 2.55 ± 1.0 × 10⁴; P < 0.05). Data are presented as mean ± s.e.m. (10 mice), and significance was calculated by an unpaired Student’s t-test. Scale bars represent 50 µm.

Figure 4. Hedgehog-induced expression of stromal Wnt signals mediates urothelial and stromal proliferation

a, Wnt2, Wnt4 and Fgf16 expression in microdissected epithelium or stroma. Stromal expression of Wnt2, Wnt4 and Fgf16 increased significantly on injury. Although Wnt4 RNA was detected in the epithelium, this expression did not increase on injury. ND, not detected. b, Laser capture microdissection of urothelial cell layers in regenerating bladder. Red, yellow, green and black lines illustrate selection and outlining of cells in basal (Bas), basal-like 1 (Bsl1), basal-like 2 (Bsl2), and intermediate (Int) layers, respectively, before microdissection. c, d, Axin2 and Shh expression in microdissected cell layers from wild-type and Gli1 mutant bladders 24 h after instillation of UTI89 or PBS. c, Expression of Axin2 increased 5.5 fold in stroma (P < 0.05), and 6.3 fold in basal (P < 0.05), 5.9 fold in basal-like 1 (P < 0.05), and 2.3 fold in basal-like 2 (P < 0.01) as compared to uninjured basal cells. Expression of Axin2 did not change significantly in basal and intermediate cells of injured Gli1 homozygous mutants as compared to basal and intermediate cells of uninjured Gli1 mutant bladder. d, Expression of Shh in cells increased 5.5 fold in basal (P < 0.01), 5.7 fold in basal-like 1 (P < 0.05), and 2.1 fold in basal-like 2 (P < 0.05) as compared to uninjured basal cells. Expression of Shh in Gli1 homozygous mutants increased 3 fold (P < 0.05) in injured as compared to uninjured basal epithelial cells. Data are presented as mean ± s.e.m., and significance was calculated by a paired Student’s t-test. Str, stroma; Umb, umbrella cells. e, Modulation of Wnt pathway activity in regenerative response to bacterial injury. Left, pharmacological reduction (indomethacin (Indo)) or augmentation (LiCl) of the Wnt signal response respectively decreases or increases bladder proliferation, with or without bacterial injury, as shown, in mice of the indicated genotype (see also Supplementary Fig. 17b, c, d). Right, tamoxifen-induced inactivation of β-catenin decreases proliferation in epithelium and stroma (CAG-CreER) or in epithelium (Shh^CreER). Data are presented as mean ± s.e.m. from 3 bladders, 2 sections each (see also Supplementary Fig. 18e, f).